Patients can die when bacteria enters the blood stream, a condition known bacteremia. This condition may occur as a result of a wound or infection, or through an invasive procedure such as surgery or an injection. Treatment of bacteremia requires hospitalization and includes the administration of intraveneous antibiotics. A life-threatening condition may develop if the bacteria are immune or resistant to the antibiotic regime used for treatment.
Once a bacterial infection reaches the blood stream, doctors only have up to 6 to 12 hours to treat the infection with the correct antibiotic before the infection will kill the patient. In those situations, understanding whether the bacteria in question is resistant to an antibiotic is critical to selecting the appropriate treatment that can save the patient. Bacterial resistance or immunity to antibiotics is becoming a growing global health problem.
In order to understand how bacterial resistance occurs, it is necessary to understand enzymes, which are complex proteins produced by living cells that speed-up or accelerate specific biochemical reactions. Enzymes are found in bacteria and it has been discovered that bacteria making, or expressing, enzymes called beta-lactamases (β-lactamases) are immune or resistant to β-lactam antibiotics.
This resistance occurs because these enzymes efficiently open up, or hydrolyze, β-lactam 4-atom rings found in major antibiotics such as penicillins, cephamycins, cephalosporins, and carbapenemes. This modification of the antibiotic molecule significantly reduces or eliminates its effectiveness as a treatment for the bacterial infection.
As a result, the ability to rapidly and accurately determine the existence of β-lactamase enzymes inside the disease causing bacteria will allow a doctor to provide an appropriate treatment that does not include beta-lactam antibiotics, and thus increase the effectiveness of the treatment and reduce potential adverse reactions from enzyme-modified beta-lactam antibiotics.
While a number of calorimetric and fluorometric compounds have been developed for the detection of β-lactamase outside the living body in a laboratory environment (in vitro), use of these compounds require external light sources to measure the presence of the enzyme. These techniques are more difficult or not possible to implement inside living creatures because the light may not reach deep tissues due to absorption of proteins such as hemoglobin, or it may generate artifacts, for example auto-fluorescence from native proteins, that may interfere with the measurement.
Detection of β-lactamases is also important to the study of biological processes, a scientific activity that is necessary to understand diseases and in developing cures. One application is the use of β-lactamase detection as a signal that a given biological process is taking place inside a living cell. A reporter system is a gene that, when it is incorporated (“transfected”) into a cell, is transformed into a protein that can be readily detected. For example, TEM-1 β-lactamase (Bla), a small (29 kDa) and monomeric enzyme, is a sensitive reporter system for detecting and for creating a spatial image of biological processes and interactions in single living cells and in tissue extracts or cultures.
Thus, a need still remains for a compound for measuring beta-lactamase enzymes that can be used inside living creatures (in vivo experiments). In addition, it would be desirable to locate exactly where the beta-lactam bacterial infection is occurring in the body by creating a spatial image showing beta-lactamase enzyme levels.
In addition, there is a need to improve the sensitivity and ease-of-use of reporter systems used for scientific research and drug development. Assay improvements generally enable new applications and reduce the cost of research and development of new pharmaceuticals or biotechnology products.
In view of the clinical importance of bacterial resistance to antibiotics, it is increasingly critical that answers be found to these problems.
Solutions to these problems have been long sought but prior developments have not taught or suggested any solutions and, thus, solutions to these problems have long eluded those skilled in the art.